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Neurons can maintain stable synaptic connections across adult life. However, the signals that regulate expression of synaptic proteins in the mature brain are incompletely understood. Here, we describe a transcriptional feedback loop between the biosynthesis and repertoire of specific phospholipids and the synaptic vesicle pool in adult Drosophila photoreceptors. Mutations that disrupt biosynthesis of a subset of phospholipids cause degeneration of the axon terminal and loss of synaptic vesicles. Although degeneration of the axon terminal is dependent on neural activity, activation of sterol regulatory element binding protein (SREBP) is both necessary and sufficient to cause synaptic vesicle loss. Our studies demonstrate that SREBP regulates synaptic vesicle levels by interacting with tetraspanins, critical organizers of membranous organelles. SREBP is an evolutionarily conserved regulator of lipid biosynthesis in non-neuronal cells; our studies reveal a surprising role for this feedback loop in maintaining synaptic vesicle pools in the adult brain., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The assembly of functional neuronal circuits requires growth cones to extend in defined directions and recognize the correct synaptic partners. Homophilic adhesion between vertebrate Sidekick proteins promotes synapse formation between retinal neurons involved in visual motion detection. We show here that Drosophila Sidekick accumulates in specific synaptic layers of the developing motion detection circuit and is necessary for normal optomotor behavior. Sidekick is required in photoreceptors, but not in their target lamina neurons, to promote the alignment of lamina neurons into columns and subsequent sorting of photoreceptor axons into synaptic modules based on their precise spatial orientation. Sidekick is also localized to the dendrites of the direction-selective T4 and T5 cells, and is expressed in some of their presynaptic partners. In contrast to its vertebrate homologs, Sidekick is not essential for T4 and T5 to direct their dendrites to the appropriate layers or to receive synaptic contacts. These results illustrate a conserved requirement for Sidekick proteins in establishing visual motion detection circuits that is achieved through distinct cellular mechanisms in Drosophila and vertebrates., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

In Drosophila, color vision and wavelength-selective behaviors are mediated by the compound eye's narrow-spectrum photoreceptors R7 and R8 and their downstream medulla projection (Tm) neurons Tm5a, Tm5b, Tm5c, and Tm20 in the second optic neuropil or medulla. These chromatic Tm neurons project axons to a deeper optic neuropil, the lobula, which in insects has been implicated in processing and relaying color information to the central brain. The synaptic targets of the chromatic Tm neurons in the lobula are not known, however. Using a modified GFP reconstitution across synaptic partners (GRASP) method to probe connections between the chromatic Tm neurons and 28 known and novel types of lobula neurons, we identify anatomically the visual projection neurons LT11 and LC14 and the lobula intrinsic neurons Li3 and Li4 as synaptic targets of the chromatic Tm neurons. Single-cell GRASP analyses reveal that Li4 receives synaptic contacts from over 90% of all four types of chromatic Tm neurons, whereas LT11 is postsynaptic to the chromatic Tm neurons, with only modest selectivity and at a lower frequency and density. To visualize synaptic contacts at the ultrastructural level, we develop and apply a "two-tag" double-labeling method to label LT11's dendrites and the mitochondria in Tm5c's presynaptic terminals. Serial electron microscopic reconstruction confirms that LT11 receives direct contacts from Tm5c. This method would be generally applicable to map the connections of large complex neurons in Drosophila and other animals., (© 2015 Wiley Periodicals, Inc.)

Histamine is an important chemical messenger that regulates multiple physiological processes in both vertebrate and invertebrate animals. Even so, how glial cells and neurons recycle histamine remains to be elucidated. Drosophila photoreceptor neurons use histamine as a neurotransmitter, and the released histamine is recycled through neighboring glia, where it is conjugated to β-alanine to form carcinine. However, how carcinine is then returned to the photoreceptor remains unclear. In an mRNA-seq screen for photoreceptor cell-enriched transporters, we identified CG9317, an SLC22 transporter family protein, and named it CarT (Carcinine Transporter). S2 cells that express CarT are able to take up carcinine in vitro. In the compound eye, CarT is exclusively localized to photoreceptor terminals. Null mutations of cart alter the content of histamine and its metabolites. Moreover, null cart mutants are defective in photoreceptor synaptic transmission and lack phototaxis. These findings reveal that CarT is required for histamine recycling at histaminergic photoreceptors and provide evidence for a CarT-dependent neurotransmitter trafficking pathway between glial cells and photoreceptor terminals.

Background: Neuronal circuits in worms, flies, and mammals are organized so as to minimize wiring length for a functional number of synaptic connections, a phenomenon called wiring optimization. However, the molecular mechanisms that establish optimal wiring during development are unknown. We addressed this question by studying the role of N-cadherin in the development of optimally wired neurite fascicles in the peripheral visual system of Drosophila., Results: Photoreceptor axons surround the dendrites of their postsynaptic targets, called lamina cells, within a concentric fascicle called a cartridge. N-cadherin is expressed at higher levels in lamina cells than in photoreceptors, and all genetic manipulations that invert these relative differences displace lamina cells to the periphery and relocate photoreceptor axon terminals into the center., Conclusions: Differential expression of a single cadherin is both necessary and sufficient to determine cartridge structure because it positions the most-adhesive elements that make the most synapses at the core and the less-adhesive elements that make fewer synapses at the periphery. These results suggest a general model by which differential adhesion can be utilized to determine the relative positions of axons and dendrites to establish optimal wiring., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The location of proteins that contribute to synaptic function has been widely studied in vertebrate synapses, far more than at model synapses of the genetically manipulable fruit fly, Drosophila melanogaster. Drosophila photoreceptor terminals have been extensively exploited to characterize the actions of synaptic genes, and their distinct and repetitive synaptic ultrastructure is anatomically well suited for such studies. Synaptic release sites include a bipartite T-bar ribbon, comprising a platform surmounting a pedestal. So far, little is known about the composition and precise location of proteins at either the T-bar ribbon or its associated synaptic organelles, knowledge of which is required to understand many details of synaptic function. We studied the localization of candidate proteins to pre- or postsynaptic organelles, by using immuno-electron microscopy with the pre-embedding method, after first validating immunolabeling by confocal microscopy. We used monoclonal antibodies against Bruchpilot, epidermal growth factor receptor pathway substrate clone 15 (EPS-15), and cysteine string protein (CSP), all raised against a fly head homogenate, as well as sea urchin kinesin (antibody SUK4) and Discs large (DLG). All these antibodies labeled distinct synaptic structures in photoreceptor terminals in the first optic neuropil, the lamina, as did rabbit anti-DPAK (Drosophila p21 activated kinase) and anti-Dynamin. Validating reports from light microscopy, immunoreactivity to Bruchpilot localized to the edge of the platform, and immunoreactivity to SUK4 localized to the pedestal of the T-bar ribbon. Anti-DLG recognized the photoreceptor head of capitate projections, invaginating organelles from surrounding glia. For synaptic vesicles, immunoreactivity to EPS-15 localized to sites of endocytosis, and anti-CSP labeled vesicles lying close to the T-bar ribbon. These results provide markers for synaptic sites, and a basis for further functional studies., ((c) 2009 Wiley-Liss, Inc.)

shibire(ts1), a temperature-sensitive mutation of the Drosophila gene encoding a Dynamin orthologue, blocks vesicle endocytosis and thus synaptic transmission, at elevated, or restrictive temperatures. By targeted Gal4 expression, UAS-shibire(ts1) has been used to dissect neuronal circuits. We investigated the effects of UAS-shibire(ts1) overexpression in Drosophila photoreceptors at permissive (19 degrees C) and restrictive (31 degrees C) temperatures. At 19 degrees C, overexpression of UAS-shi(ts1) causes decelerated phototransduction and reduced neurotransmitter release. This phenotype is exacerbated with dark adaptation, age and in white mutants. Photoreceptors overexpressing UAS-shibire(ts1) contain terminals with widespread vacuolated mitochondria, reduced numbers of vesicles and bundled microtubules. Immuno-electron microscopy reveals that the latter are dynamin coated. Further, the microtubule phenotype is not restricted to photoreceptors, as UAS-shibire(ts1) overexpression in lamina cells also bundles microtubules. We conclude that dynamin has multiple functions that are interrupted by UAS-shibire(ts1) overexpression in Drosophila photoreceptors, destabilizing their neural communication irreversibly at previously reported permissive temperatures.

As a neuron differentiates, it adopts a suite of features specific to its particular type. Fly photoreceptors are of two types: R1-R6, which innervate the first optic neuropile, the lamina; and R7-R8, which innervate the second, the medulla. Photoreceptors R1-R6 normally have large light-absorbing rhabdomeres, express Rhodopsin1, and have synaptic terminals that innervate the lamina. In Drosophila melanogaster, we used the yeast GAL4/UAS system to drive exogenous expression of the transcription factor Runt in subsets of photoreceptors, resulting in aberrant axonal pathfinding and, ultimately, incorrect targeting of R1-R6 synaptic terminals to the medulla, normally occupied by terminals from R7 and R8. Even when subsets of their normal R1-R6 photoreceptor inputs penetrate the lamina, to terminate in the medulla, normal target cells within the lamina persist and maintain expression of cell-specific markers. Some R1-R6 photoreceptors form reciprocal synaptic inputs with their normal lamina targets, whereas supernumerary terminals targeted to the medulla also form synapses. At both sites, tetrad synapses form, with four postsynaptic elements at each release site, the usual number in the lamina. In addition, the terminals at both sites are invaginated by profiles of glia, at organelles called capitate projections, which in the lamina are photoreceptor sites of vesicle endocytosis. The size and shape of the capitate projection heads are identical at both lamina and medulla sites, although those in the medulla are ectopic and receive invaginations from foreign glia. This uniformity indicates the cell-autonomous determination of the architecture of its synaptic organelles by the presynaptic photoreceptor terminal.

Drosophila vision is mediated by inputs from three types of photoreceptor neurons; R1-R6 mediate achromatic motion detection, while R7 and R8 constitute two chromatic channels. Neural circuits for processing chromatic information are not known. Here, we identified the first-order interneurons downstream of the chromatic channels. Serial EM revealed that small-field projection neurons Tm5 and Tm9 receive direct synaptic input from R7 and R8, respectively, and indirect input from R1-R6, qualifying them to function as color-opponent neurons. Wide-field Dm8 amacrine neurons receive input from 13-16 UV-sensing R7s and provide output to projection neurons. Using a combinatorial expression system to manipulate activity in different neuron subtypes, we determined that Dm8 neurons are necessary and sufficient for flies to exhibit phototaxis toward ultraviolet instead of green light. We propose that Dm8 sacrifices spatial resolution for sensitivity by relaying signals from multiple R7s to projection neurons, which then provide output to higher visual centers.

Ectopic expression of the retinal determination gene eyeless (ey) induces the formation of supernumerary eyes on antennae, legs, wings, and halteres. These ectopic eyes form ommatidia that contain photoreceptors and accessory cells and respond to light. Here, we demonstrate that ectopic eyes on antennae and legs extend axonal projections to the central nervous system. Furthermore, electroretinograms and morphological evidence indicate that the photoreceptor axons of at least the antennal ectopic eyes can form completely constituted ectopic synapses with foreign postsynaptic elements and suggest that transmission at these sites may be functional. However, the ectopic axons do not connect to their correct optic lobe targets and do not project deeply into the neuropile, but rather form synapses at superficial positions in the neuropils. By means of confocal and electron microscopy we show that these ectopic synapses resemble normal synapses, albeit with some distinct morphological differences. Our data strongly suggest that the developmental programs controlling photoreceptor synaptogenesis and visual map formation depend to a considerable extent on presynaptic and thus photoreceptor-autonomous steps. Our data also suggest that photoreceptor axon projections and the establishment of the highly stereotypical neural circuitry in the optic lobe, the normal target neuropil, may depend on target-specific cues that appear to be absent from the antennal lobe and thoracic ganglion.

Histamine, a ubiquitous aminergic messenger throughout the body, also serves as a neurotransmitter in both vertebrates and invertebrates. In particular, the photoreceptors of adult arthropods use histamine, modulating its release to signal increases and decreases in light intensity. Strong evidence from various arthropod species indicates that histamine is synthesized and stored in photoreceptors, undergoes Ca-dependent release, inhibits postsynaptic interneurons by gating Cl channels, and is then recycled. In Drosophila, the synthetic enzyme, histidine decarboxylase, and the subunits of the histamine-gated chloride channel have been cloned. Possible histamine transporters at synaptic vesicles and for reuptake remain elusive. Indeed, the mechanisms that remove histamine from the synaptic cleft, and that help terminate histamine's action, are unexpectedly complex, their details remaining unresolved. A major pathway in Drosophila, and possibly other arthropod species, is by conjugation of histamine to beta-alanine to form carcinine in adjacent glia. This conjugate then returns to the photoreceptors where it is hydrolysed to liberate histamine, which is then loaded into synaptic vesicles. Evidence from other species suggests that direct reuptake of histamine into the photoreceptors may also occur. Light depolarizes the photoreceptors, causing histamine release and postsynaptic inhibition; dimming hyperpolarizes the photoreceptors, causing a decrease in histamine release and an "off" response in the postsynaptic cell. Further pursuit of histamine's action at these highly specialized synapses should lead to an understanding of how they signal minute changes in presynaptic membrane potential, how they reliably extract signals from noise, and how they adapt to a wide range of presynaptic membrane potentials.

Specifying synaptic partners and regulating synaptic numbers are at least partly activity-dependent processes during visual map formation in all systems investigated to date . In Drosophila, six photoreceptors that view the same point in visual space have to be sorted into synaptic modules called cartridges in order to form a visuotopically correct map . Synapse numbers per photoreceptor terminal and cartridge are both precisely regulated . However, it is unknown whether an activity-dependent mechanism or a genetically encoded developmental program regulates synapse numbers. We performed a large-scale quantitative ultrastructural analysis of photoreceptor synapses in mutants affecting the generation of electrical potentials (norpA, trp;trpl), neurotransmitter release (hdc, syt), vesicle endocytosis (synj), the trafficking of specific guidance molecules during photoreceptor targeting (sec15), a specific guidance receptor required for visual map formation (Dlar), and 57 other novel synaptic mutants affecting 43 genes. Remarkably, in all these mutants, individual photoreceptors form the correct number of synapses per presynaptic terminal independently of cartridge composition. Hence, our data show that each photoreceptor forms a precise and constant number of afferent synapses independently of neuronal activity and partner accuracy. Our data suggest cell-autonomous control of synapse numbers as part of a developmental program of activity-independent steps that lead to a "hard-wired" visual map in the fly brain.

Reliable estimates of the quantum size in histaminergic neurons are not available. We have exploited two unusual opportunities in the fly's (Drosophila melanogaster) visual system to make such determinations for histaminergic photoreceptor synapses: 1) the possibility to microdissect successively from whole fly heads freeze-dried in acetone: the compound eyes; the first optic neuropils, or lamina; and the rest of the brain; and 2) the uniform sheaves of lamina synaptic terminals of photoreceptors R1-R6. We used this organization to count scrupulously the numbers of 30-nm synaptic vesicles from electron micrographs of R1-R6 profiles, and from microdissections we determined the regional contents of histamine in the compound eye, lamina, and central brain. Total head histamine averages 1.98 ng of which 9% was lost after freeze-drying in acetone and a further 28% after the brain was microdissected. Of the remainder, 71% was in the eye and lamina. Assuming that histamine loss from the tissue occurred mostly by diffusion evenly distributed among all regions, the overall lamina content of the head would be 0.1935 ng before dissection. From published values for the volumes of the brain's compartments, the computed regional concentrations of histamine are highest in the lamina (4.35 mM) because of the terminals of R1-R6. The concentration in the retina is approximately 13% that in the lamina, suggesting that most histamine is vesicular. There are approximately 43,500 +/- 7,400 (SD) synaptic vesicles per terminal and, if all histamine is allocated equally and exclusively among these, the vesicle contents would be 858 +/- 304 x 10(-21) moles or approximately 5,000 +/- 1,800 (SD) molecules at an approximate concentration of 670 mM. These values are compared with the vesicle contents at synapses using acetylcholine and catecholamines.

Retrieval of synaptic vesicles from the membrane of neurons is crucial to maintain normal rates of neurotransmitter release. Photoreceptor terminals of the fly's eye release neurotransmitter in a tonic manner. They therefore rely heavily on vesicle regeneration. Null mutations in endophilin (endo) block clathrin-mediated endocytosis at the Drosophila neuromuscular junction, where previous analysis of hypomorphic mutations has suggested a function for Endophilin (Endo) before vesicle fission, during membrane bending. Here, at fly photoreceptor synapses, we show that Endo is localized to synaptic vesicles at sites of endocytosis that are glial invaginations called capitate projections, and that when the photoreceptor synapses lack Endo they are impaired in their ability to release neurotransmitter. Detailed ultrastructural analysis of endo null mutant photoreceptor synapses fails to reveal a defect at early stages of vesicle reformation but, instead, reveals an accumulation of clusters of electron-dense, apparently nonfunctional, late endocytotic vesicles. Using dynamin;endo double-mutant photoreceptors, we provide further evidence that ultimately the function of Endophilin is required late in endocytosis, allowing vesicles to progress through the synaptic vesicle cycle.

Photoreceptor neurons (R cells) in the Drosophila visual system elaborate a precise map of visual space in the brain. The eye contains some 750 identical modules called ommatidia, each containing eight photoreceptor cells (R1-R8). Cells R1-R6 synapse in the lamina; R7 and R8 extend through the lamina and terminate in the underlying medulla. In a screen for visual behavior mutants, we identified alleles of flamingo (fmi) that disrupt the precise maps elaborated by these neurons. These mutant R1-R6 neurons select spatially inappropriate targets in the lamina. During target selection, Flamingo protein is dynamically expressed in R1-R6 growth cones. Loss of fmi function in R cells also disrupts the local pattern of synaptic terminals in the medulla, and Flamingo is transiently expressed in R8 axons as they enter the target region. We propose that Flamingo-mediated interactions between R-cell growth cones within the target field regulate target selection.

A protein required to localize mitochondria to Drosophila nerve terminals has been identified genetically. Photoreceptors mutant for milton show aberrant synaptic transmission despite normal phototransduction. Without Milton, synaptic terminals and axons lack mitochondria, although mitochondria are numerous in neuronal cell bodies. In contrast, synaptic vesicles continue to be transported to and concentrated at synapses. Milton protein is associated with mitochondria and is present primarily in axons and synapses. A likely explanation of the apparent trafficking defect is offered by the coimmunoprecipitation of Milton and kinesin heavy chain. Transfected into HEK293T cells, Milton induces a redistribution of mitochondria within the cell. We propose that Milton is a mitochondria-associated protein required for kinesin-mediated transport of mitochondria to nerve terminals.

In Drosophila melanogaster, ebony and tan, two cuticle melanizing mutants, regulate the conjugation (ebony) of beta-alanine to dopamine or hydrolysis (tan) of the beta-alanyl conjugate to liberate dopamine. beta-alanine biosynthesis is regulated by black. ebony and tan also exert unexplained reciprocal defects in the electroretinogram, at ON and OFF transients attributable to impaired transmission at photoreceptor synapses, which liberate histamine. Compatible with this impairment, we show that both mutants have reduced histamine contents in the head, as measured by HPLC, and have correspondingly reduced numbers of synaptic vesicles in their photoreceptor terminals. Thus, the histamine phenotype is associated with sites of synaptic transmission at photoreceptors. We demonstrate that when they receive microinjections into the head, wild-type Sarcophaga bullata (in whose larger head such injections are routinely possible) rapidly (<5 sec) convert exogenous [3H]histamine into its beta-alanine conjugate, carcinine, a novel metabolite. Drosophila tan has an increased quantity of [3H]carcinine, the hydrolysis of which is blocked; ebony lacks [3H]carcinine, which it cannot synthesize. Confirming these actions, carcinine rescues the histamine phenotype of ebony, whereas beta-alanine rescues the carcinine phenotype of black;tan double mutants. The equilibrium ratio between [3H]carcinine and [3H]histamine after microinjecting wild-type Sarcophaga favors carcinine hydrolysis, increasing to only 0.5 after 30 min. Our findings help resolve a longstanding conundrum of the involvement of tan and ebony in photoreceptor function. We suggest that reversible synthesis of carcinine occurs in surrounding glia, serving to trap histamine after its release at photoreceptor synapses; subsequent hydrolysis liberates histamine for reuptake.

The first neuropile, or lamina, of the fly's optic lobe comprises a model set of identified neurons that are arrayed in cylindrical modules, called cartridges. The cartridge acquires adult form only in the second half of the fly's pupal life. All cells are by then correctly located within each of the lamina's cartridges (Drosophila, Musca), becoming invested by glial cells after 75% of pupal development (P + 75%). In adult cartridges, two lamina cells, L1 and L2, receive input from photoreceptor terminals R1-R6, at so-called tetrad synapses that form in the pupa when these cells' dendrites contact R1-R6. Single-section electron microscopy (EM, Drosophila) and serial-EM reconstructions of L1 and L2 (Musca) reveal relationships between the morphogenesis of L1/L2 dendrites and the formation of tetrads. Neurite outgrowth is initially (P + 55%) random and neurites are unbranched; many neurites invaginate surrounding terminals of R1-R6 but, later, embrace the outer surfaces of these. The maximum profusion of neurites at P + 74% coincides with peak numbers of nascent tetrads; neurites then branch vertically, in the lamina's depth. Later, neurites failing to reach R1-R6's outer surfaces regress. Down the length of their axons, L1 and L2's neurites initially form a random sequence, L1 partnering L1 as often as L2, etc., but beginning at P + 74%, L1 partners L2, and L2 partners L1, with progressive strictness. L1 has more neurites overall than L2. These observations are consistent with the following hypotheses: a neurite only survives if it contacts a presynaptic site; a synapse only survives if it progressively acquires the appropriate number and combination of postsynaptic neurites, culminating in a tetrad; an interaction exists between the neurites of L1 and L2, so that the growth of one respects the pattern of growth of the other.

Many invertebrates have supplementary extraocular photoreceptors that often are implicated in circadian rhythms. An extraretinal group of candidate photoreceptors in the fruit fly, Drosophila melanogaster, has been revealed previously at the posterior margin of the compound eye by using a photoreceptor-specific monoclonal antibody (Hofbauer and Buchner [1989] Naturwissen 76:335-336), but it never has been characterized. Here, we report the fine structure of this cell cluster reported by Hofbauer and Buchner, which is called "eyelet," as well as the further candidacy of their visual pigment and neurotransmitter. Eyelet forms a specialized, pigmented organ with cells that have numerous microvilli arranged into coherent rhabdomeres. The presence of rhabdomeric microvilli is a defining feature of a photoreceptor, reported here for the first time in eyelet. The rhabdomeres exhibit Rh6 opsin-like immunoreactivity, which provides evidence that the photoreceptors are functional: they fail to immunostain with antibodies against NINAE (Rh1), Rh4, or Rh5. The photoreceptors have been shown previously to exhibit histamine-like immunoreactivity, but they also stain with a monoclonal antiserum raised against Drosophila choline acetyltransferase (ChAT), suggesting that the photoreceptors not only may contain histamine but also can synthesize acetylcholine. A ChAT-immunoreactive axon bundle originating from eyelet terminates in the cortex of the anterior medulla. This bundle also is seen with reduced silver stains. Electron microscopic examination revealed four axon profiles of similar size in this bundle, indicating that eyelet contains at least four photoreceptors. The pathway of eyelet's axon bundle coincides with the precocious pathway of Bolwig's nerve that arises from the larval organ of sight. The origin and possible function of eyelet are discussed., (Copyright 1999 Wiley-Liss, Inc.)

The visual system of the fly's compound eye undergoes a number of cyclical day/night changes that have a circadian basis. Such responses are seen in the synaptic terminals of the photoreceptors and in their large monopolar-cell interneurons in the first optic neuropile, or lamina. These changes include, in the photoreceptor terminals, rhythms in the numbers of synapses and the vertical migration of screening pigment; and, in the monopolar cells L1 and L2, a rhythm in the transients of the electroretinogram and in the cyclical swelling of L1 and L2 lamina axons, as well as of the epithelial glia that surround these. Some of these changes are seen in both the housefly and the fruit fly, but the time-course of such changes differs between the two species. Many of the changes are influenced by the injection of various transmitter candidates, in a direction that can be reconciled with the possibility of normal endogenous release of two substances, 5HT from the neurites of 5HT-immunoreactive neurons, and pigment dispersing factor peptide from the neurites of PDH cells. Consistent with this interpretation, the immunoreactive varicosities of PDH cells exhibit size changes attributable to their cyclical release of peptide, or to its cyclical synthesis and/or transport from the PDH cell somata. Thus, neurotransmitter substances not only have rapid electrophysiological actions in the optic lobe, but also longer-lasting, presumably indirect, neuromodulatory actions, which are manifest as structural changes among the lamina's neurons and synapses. These actions involve an interplay between aminergic and peptidergic systems, but the exact role and especially the site of action of each has still to be elucidated.

Various organelles in the lamina terminals of housefly photoreceptors exhibit daily rhythms having a circadian basis. These include changes in the numbers of photoreceptor tetrad and L2 feedback synapses, and longitudinal movements of screening pigment. Circadian information has previously been suggested to spread from the clock to the lamina via widefield cells expressing either 5-hydroxytryptamine or pigment-dispersing hormone-like immunoreactivity. We examined the action of these neuromodulators, and other candidate neurotransmitters, 4 h after injecting either the transmitter or a control into the medulla. We counted electron microscope profiles of organelles that normally exhibit circadian changes, and two types of invagination into photoreceptor terminals, capitate projections and inter-receptor invaginations. No single substance mediated the changes observed. Injected pigment-dispersing hormone peptide decreased the number of pigment granules, implicating this peptide in screening pigment migration, but produced no changes in synapse-related organelles. alpha-Aminobutyric acid exclusively decreased the number of L2 feedback synapses. Responses to other transmitters were specific, and often large, but generally not statistically significant. Histamine, for example, may decrease the number of tetrads, possibly by direct autoregulation. The results suggest that there is likely to be more than one effector in the circadian pathways to the lamina.

The molecular structure of insect gap junctions differs from that in vertebrates, and in Drosophila is possibly encoded by the shaking B (= Passover) locus. shaking B2 is a null allele that acts in the nervous system. In the shakB2 mutant, one site of action are gap junctions between photoreceptor terminals in the cartridges of the lamina, beneath the compound eye, which we assayed from the number of close-apposition profiles in thin-section EM. The number of profiles in the Canton-S (C-S) wild type is about 0.5 per cartridge per section in distal and mid-lamina depths, and significantly less, about one quarter this value, closer to the brain, in the proximal lamina. In shakB2, there are fewer profiles, approximately one quarter the number of appositions in distal and mid-lamina depths as in C-S, and their number does not differ significantly from those at the proximal depth in either the mutant or wild type. Thus mutant action is associated with a reduced number of appositions at distal and mid-lamina depths. We propose that R1-R6 gap junctions are partitioned into at least two strata, proximal and distal, and that two populations of gap junctions exist, one extending throughout the lamina that does not require shakB, and a second at distal and mid-depth levels, which does. The number of gap junctions is reduced in mutant shakB2, and surviving appositions at distal and middle lamina depths possibly have wider clefts than in C-S. Gap junctions are reduced equally between all R1-R6 terminals, so the two different types of junction proposed, shakB2- and non-shakB2-dependent, can apparently express in a single receptor terminal.

We have examined the effects of the insect ecdysteroid, 20-hydroxyecdysone, on the differentiation of neuronal and non-neuronal elements in the developing adult visual system, using in vitro methods in Drosophila. We examined the differentiation of early neuronal markers in the presence and absence of 1 microgram/ml 20-hydroxyecdysone. Immunoreactivity to 22C10, a marker of an early neuronal antigen, as well as to the photoreceptor-specific antibody 24B10, suggests that differentiation of neuronal and photoreceptor antigens does not require 20-hydroxyecdysone. In eye-discs cultured from animals 5 hours after the white prepupa (P + 5), ommochrome pigmentation first appeared after 2 days in 1 microgram/ml 20-hydroxyecdysone, but cultures lacked pigmentation without 20-hydroxyecdysone. Our culture conditions failed to support the formation of the second screening pigment, drosopterins, even with 20-hydroxyecdysone. Eye discs from P + 5 also formed lenses and interommatidial bristles in culture when 20-hydroxyecdysone was added but not in cultures devoid of the hormone. The differentiation of synaptotagmin and the elongation of extending photoreceptor neurites from eye disc fragments both occur in the absence of 20-hydroxyecdysone in cultures, but adding the hormone increased average neurite length. The threshold for enhanced neurite length was less than 125 ng/ml 20-hydroxyecdysone. Eye-disc cultures also developed immunoreactivity to histamine, the photoreceptor transmitter, from synthesis not re-uptake, in both the presence and in the absence of 20-hydroxyecdysone. These findings suggest that photoreceptor axons may be able to release transmitter in vivo both when they grow into the optic lobe and during the subsequent events in synapse formation.

Screening pigment granules occur in the synaptic terminals of photoreceptors in the fly's (Musca domestica, L.) compound eye. The granules resemble ommochrome granules in the overlying photoreceptor cell body. There are also two types of invagination into receptor terminals: capitate projections (from glial cells) and invaginations from neighboring receptor terminals. The number of profiles of these organelles in the first optic neuropile, the lamina, have been counted using single-section quantitative electron microscopic methods. Pigment granules are concentrated proximally in the terminal, toward the brain. The numbers change, increasing during the night (1 h after lights off) up to values more than twice the number 1 h after lights on, apparently by longitudinal migration of granules from the cell body into the terminal. Files entrained to day/night conditions and then held under constant darkness continue to exhibit changes in the numbers of profiles. Even though overall there were 80-90% fewer granule profiles than under day/night conditions, the numbers attained a peak many times higher at the end of the subjective day. Thus, the changes are endogenous, showing circadian rhythmicity. Although their significance is unknown, these changes parallel previously described circadian rhythms in the receptor terminals and their lamina monopolar-cell targets. The invaginations from receptor terminals were more numerous under day/night conditions than under constant darkness, and cycled in constant darkness, peaking at the end of subjective night. Capitate projections, by contrast, failed to change significantly under the experimental conditions analyzed, a lack of responsiveness they share with photoreceptor tetrad synapses.

The short-term influence of visual experience was studied in the first-order tetrad synapses of the first optic neuropil, or lamina, underlying the compound eye in the housefly (Musca domestica). We report the effects of single light reversals, in which flies reared in constant darkness were exposed to light or those in constant light were exposed to darkness, on this defined population of synapses. The synaptic population was characterized by quantitative electron microscopic methods using three parameters: (i) the number of synaptic contacts per photoreceptor terminal; (ii) the size of these synapses, given by the mean platform width of their presynaptic ribbons; and (iii) the proportion (up to approximately 20%) of small synapses-those having a ribbon platform less than 0.17 micron wide. The effects of light exposures in flies reared in constant darkness include decreased mean synaptic size and increased numbers of synapses. These effects are seen in flies of all ages (to 10 days). Long light exposures (2-6 h) are much less effective than short exposures (down to 20 s), with maximum effects obtained at 15 min (up to 45% more synapses). Small synapses are reasoned to be new junctions formed only recently; the decreased mean synaptic size and the increased number of synapses seen after short light exposures are both interpreted to result from a recent burst of synaptogenesis in the adult lamina. The effects of dark exposure in flies reared in constant light are the reciprocal of those seen in dark-reared flies exposed to light, but they are less pronounced. Although the function of such changes is not yet known, they may form part of the light adaptation mechanism of the photoreceptor, and occur along with a redistribution of other organelles involving membrane invaginations into its terminal. These changes occur against a background trend for control flies reared under constant conditions to have fewer, larger synapses with increasing age up to 10 days, an effect that is most pronounced in constant darkness, when synaptic number decreases by 21% and size increases by 13%.

1. Photoreceptor terminals in the flies Musca domestica and Drosophila melanogaster have been reconstructed in three dimensions from serial EM to reveal the surface distributions of afferent tetrad synapses. 2. The terminals are cylindrical and surround two target cells; they have synaptic sites distributed along their length and around their circumference, except for a strip along the face that lies furthest away from the target cells. 3. Over their inner faces, the terminals have presynaptic sites that are distributed evenly. 4. The distribution of sites in maps plotted from reconstructed membrane surfaces was examined by quadrat analyses. The frequency of sites per quadrat division was not Poissonian, i.e. was non-random. Thus, some form of site selection must exist during synaptogenesis. 5. The sites were shown by variance ratio analysis to be regular (evenly dispersed, not clustered). This suggests that some form of interaction exists, so as to reduce the probability that a synapse will form close to an already existing synaptic site. 6. Distances between nearest-neighbour pairs of synapses had a closest minimum spacing of about 0.8 micron in Musca that was violated by about 5% of pairs, whereas the corresponding distances were about 0.2 micron shorter in Drosophila, which had 13% of pairs situated closer together than 0.8 micron. 7. During synaptogenesis, either initially in the pupa or later in the adult, the probability that a synapse will form is therefore effectively zero within these distances from an existing synaptic site, perhaps through an inhibitory influence exerted by the latter. The nearest-neighbour distances are normally distributed. 8. Unlike the distribution of presynaptic sites, the distribution of postsynaptic sites over the surfaces of the dendrites of the target cells is not even. Although not studied in detail, the corresponding nearest-neighbour distances are much smaller, as little as 0.1 micron. Thus the wider spacing seen between sites over the receptor terminals is a function of the presynaptic cells, and not of their postsynaptic partners, and implies the existence of interactions between synaptic sites.

When a housefly, Musca domestica, is subject to cold exposure (0 degrees C for 24 hr), a number of obvious changes are seen in the first optic neuropil, or lamina, beneath the compound eye. In particular, the number of afferent photoreceptor synapses declines by about 30%. This loss is dramatically restored after warm recovery at 23 degrees C for 24 hr. Synapses disappear at an average rate of 2-3/hr during cold exposure and reappear at a maximal rate of more than 20/hr during the first 2 hr of warm recovery. Thereafter their number temporarily overshoots control values, to increase at 6 hr of warm recovery to 60% above their cold-exposed minimum. The number subsequently returns more or less to normal. These changes demonstrate the lability of synaptic sites under these conditions, with individual sites forming and disappearing rapidly. The changes also interrupt the close correlation between synaptic number and the surface area of the receptor terminal, a correlation that normally conserves synaptic spacing density. The density is preserved during cold exposure but increases during warm recovery at a time when the addition of newly formed synapses exceeds the slower increase in receptor terminal size.

Photoreceptors of the fly's compound eye generally show no very obvious daily or circadian rhythms, a lack which prompted us to examine whether their function might be regulated not in the retina, but at the site of transmission in the first visual neuropile, or lamina. Here, photoreceptor terminals (R1-R6) are reciprocally interconnected with one class of lamina monopolar cell, L2: L2 receives input from R1-R6 at so-called tetrad synapses, and in turn is presynaptic to R1-R6 at feedback synapses. We have calculated the mean frequencies of these synaptic profiles in electron micrographs of single lamina sections. L2 feedback synapses were more numerous at night than during the day, whereas the number of tetrads showed only small modulations between day and night. These changes persisted amongst feedback synapses in flies held in constant darkness, and are thus circadian. In contrast to the slow modulations during a 24 h cycle, the number of L2 feedback synapses after 1 h light pulse in flies held in constant darkness showed no clear change, whereas it increased the number of tetrad profiles. These findings support the occurrence of cyclical daily and circadian changes amongst the two lamina synaptic populations, with tetrads showing rather weak modulations in frequency, but more pronounced responses to the light pulse than feedback synapses.